Optimizing the formation of the acquired enamel pellicle in vitro for proteomic analysis

Saliva is the major contributor for the protein composition of the acquired enamel pellicle (AEP), a bacteria-free organic layer formed by the selective adsorption of salivary proteins on the surface of the enamel. However, the amount of proteins that can be recovered is even smaller under in vitro condition, due to the absence of continuous salivary flow. Objective: This study developed an in vitro AEP protocol for proteomics analysis using a new formation technique with different collection solutions. Methodology: 432 bovine enamel specimens were prepared (4x4 mm) and divided into four groups (n=108). Unstimulated saliva was provided by nine subjects. The new AEP formation technique was based on saliva resupply by a new one every 30 min within 120 minutes at 37ºC under agitation. AEP was collected using an electrode filter paper soaked in the collection solutions according with the group: 1) 3% citric acid (CA); 2) 0.5% sodium dodecyl sulfate (SDS); 3) CA followed by SDS (CA+SDS); 4) SDS followed by CA (SDS+CA). The pellicles collected were processed for analysis through LC-ESI-MS/MS technique. Results: A total of 55 proteins were identified. The total numbers of proteins identified in each group were 40, 21, 28 and 41 for the groups CA, SDS, CA+SDS and SDS+CA, respectively. Twenty-three typical AEP proteins were identified in all groups, but Mucin was only found in CA and CA+SDS, while three types of PRP were not found in the SDS group. Moreover, a typical enamel protein, Enamelin, was identified in the CA+SDS group only. Conclusion: The new technique of the in vitro AEP formation through saliva replacement was essential for a higher number of the proteins identified. In addition, considering practicality, quantity and quality of identified proteins, citric acid seems to be the best solution to be used for collection of AEP proteins

Updating the role of matrix metalloproteinases in mineralized tissue and related diseases

Bone development and healing processes involve a complex cascade of biological events requiring well-orchestrated synergism with bone cells, growth factors, and other trophic signaling molecules and cellular structures. Beyond health processes, MMPs play several key roles in the installation of heart and blood vessel related diseases and cancer, ranging from accelerating metastatic cells to ectopic vascular mineralization by smooth muscle cells in complementary manner. The tissue inhibitors of MMPs (TIMPs) have an important role in controlling proteolysis. Paired with the post-transcriptional efficiency of specific miRNAs, they modulate MMP performance. If druggable, these molecules are suggested to be a platform for development of “smart” medications and further clinical trials. Thus, considering the pleiotropic effect of MMPs on mammals, the purpose of this review is to update the role of those multifaceted proteases in mineralized tissues in health, such as bone, and pathophysiological disorders, such as ectopic vascular calcification and cancer

Metabolic effect of low fluoride levels in the islets of NOD mice: integrative morphological, immunohistochemical, and proteomic analyses

Fluoride (F) has been widely used to control dental caries, and studies suggest beneficial effects against diabetes when a low dose of F is added to the drinking water (10 mgF/L). Objectives: This study evaluated metabolic changes in pancreatic islets of NOD mice exposed to low doses of F and the main pathways altered by the treatment. Methodology: In total, 42 female NOD mice were randomly divided into two groups, considering the concentration of F administered in the drinking water for 14 weeks: 0 or 10 mgF/L. After the experimental period, the pancreas was collected for morphological and immunohistochemical analysis, and the islets for proteomic analysis. Results: In the morphological and immunohistochemical analysis, no significant differences were found in the percentage of cells labelled for insulin, glucagon, and acetylated histone H3, although the treated group had higher percentages than the control group. Moreover, no significant differences were found for the mean percentages of pancreatic areas occupied by islets and for the pancreatic inflammatory infiltrate between the control and treated groups. Proteomic analysis showed large increases in histones H3 and, to a lesser extent, in histone acetyltransferases, concomitant with a decrease in enzymes involved in the formation of acetyl-CoA, besides many changes in proteins involved in several metabolic pathways, especially energy metabolism. The conjunction analysis of these data showed an attempt by the organism to maintain protein synthesis in the islets, even with the dramatic changes in energy metabolism. Conclusion: Our data suggests epigenetic alterations in the islets of NOD mice exposed to F levels comparable to those found in public supply water consumed by humans

Do commercial whitening dentifrices increase enamel erosive tooth wear?

Objective: This in vitro study evaluated the effect of commercial whitening dentifrices on erosive tooth wear (ETW) of bovine enamel samples, in comparison with commercial regular dentifrices. Methodology: Sixty bovine crowns were embedded in acrylic resin, polished and then had their baseline profile determined. They were randomly assigned to 5 groups (n=12/group), according to the type of commercial dentifrice to be tested: GI – Crest Anti-cavity Regular; GII – Crest 3D White; GIII – Colgate Total 12 Clean Mint; GIV – Colgate Optic White; GV – Placebo (negative control, fluoride-free dentifrice). The samples were submitted to daily erosive and abrasive challenges for 3 days. The erosive challenges were performed 3 times a day by immersing the specimens in 0.1% citric acid solution (pH 2.5) for 90 s. Each day after the first and last erosive challenges, the specimens were subjected to the abrasive challenge for 15 s, using a toothbrushing machine (Biopdi, São Carlos, SP, Brazil), soft toothbrushes and slurry (1:3 g/ml) of the tested toothpastes (1.5 N). The specimens were kept in artificial saliva between the challenges. The final profile was obtained and the ETW (µm) was calculated. Data were analyzed by Kruskal-Wallis and Dunn’s tests (p<0.05). Results: All dentifrices tested significantly reduced the enamel wear in comparison with the Placebo, except GIII. The median (95% CI) ETW was 1.35 (1.25-1.46)bc for GI, 1.17 (1.01-1.34)cd for GII, 1.36 (1.28-1.45)ab for GIII, 1.08 (1.04-1.14)d for GIV and 2.28 (2.18-2.39)a for GV. Conclusion: When dentifrices from the same manufacturer were compared, the whitening dentifrices led to similar or less wear than the regular ones

Acquired enamel pellicle protects gastroesophageal reflux disease patients against erosive tooth wear

Abstract The objective of this study was to compare the protein profile of the acquired enamel pellicle (AEP) formed in vivo in patients with or without gastroesophageal reflux disease (GERD), and with or without erosive tooth wear (ETW). Twenty-four volunteers were divided into 3 groups: 1) GERD and ETW; 2) GERD without ETW; and 3) control (without GERD). The AEP formed 120 min after prophylaxis was collected from the lingual/palatal surfaces. The samples were subjected to mass spectrometry (nLC-ESI-MS/MS) and label-free quantification by Protein Lynx Global Service software. A total of 213 proteins were identified, or 119, 92 and 106 from each group, respectively. Group 2 showed a high number of phosphorylated and calcium-binding proteins. Twenty-three proteins were found in all the groups, including 14-3-3 protein zeta/delta and 1-phosphatidylinositol. Several intracellular proteins that join saliva after the exfoliation of oral mucosa cells might have the potential to bind hydroxyapatite, or participate in forming supramolecular aggregates that bind to precursor proteins in the AEP. Proteins might play a central role in protecting the dental surface against acid dissolution

Análise proteômica da película adquirida do esmalte e saliva em pacientes com câncer de cabeça e pescoço submetidos à radioterapia

Salivary glands are affected during radiotherapy in the head and neck region, leading to reduction in salivary flow and changing saliva composition. In this thesis, comprising 6 articles, we evaluated changes in the proteomic profile of the acquired enamel pellicle (AEP) and saliva in patients with head and neck cancer (HNC) submitted to radiotherapy, to search for therapeutic strategies and prognosis biomarkers. In the first two articles the protocols for standardization of proteomic analysis of saliva and collection of AEP are described. In articles 3, 4 and 5, HNC patients had their AEP and saliva (unstimulated and stimulated saliva) collected before (BRT), during (2-5 weeks; DRT) and after (3-4 months; ART) radiotherapy. Saliva and AEP were also collected from healthy patients (control; C). Proteins were extracted and processed for label-free proteomics. Salivary flows were also evaluated. In total, 1,055 proteins were identified in the unstimulated saliva, among which 47 were common to all groups, while 86, 86, 286 and 395 were exclusively found in C, BRT, DRT and ART, respectively. Remarkably, alpha-enolase was increased 35-fold DRT compared with BRT, while proline-rich proteins (PRPs) were decreased. ART there was a 16-fold increase in scaffold attachment factor-B1 and a 3-fold decrease in alpha-enolase and cystatins. When compared with C, salivary proteins of BRT patients showed increases in cystatin-C, lysozyme C, histatin-1 and PRPs (article 3). Significant differences were observed between stimulated and unstimulated salivary flows for C and BRT (p>0.001), but not for DRT and ART. Proteins involved with apoptosis and antibacterial function were decreased in stimulated saliva in comparison to unstimulated saliva DRT and ART (article 4). In the AEP, statherin was increased more than 9-fold and hemoglobins were increased more than 5-fold DRT compared to BRT, while lactotransferrin, PRPs cystatins, neutrophil defensins and histatin-1 were decreased. ART, lactotransferrin and isoforms of histones were increased, while statherin and alpha-amylase were decreased. MOAP-1 was exclusively found ART compared to BRT. When compared to Control, AEP of patients BRT showed an increase in proteins related to the perception of bitter taste, mucin-7 and alpha-amylases, while cystatin-S was decreased (article 5). In article 6, we evaluated the protective effect AEP formed in vitro for different times on enamel, as well as its engineering with statherin peptide (StatpSpS), against initial erosion. AEP provided almost instant protection, with formation times as short as 1 min protecting the native enamel against erosion, and longer formation times did not improve the protection. StatpSpS by itself provided similar protection as the AEP. In conclusion, both HNC and radiotherapy remarkably alter the proteome of saliva and AEP. The unstimulated salivary flow is the best alternative to search for biomarkers. Monitoring alpha-enolase levels in unstimulated saliva DRT and MOAP-1 in AEP ART might be possible strategies to predict the efficacy of radiotherapy. Our results provide important information for designing more effective dental products for these patients and contribute for a better understanding of the progressive changes in salivary proteins induced by radiotherapy and the protective roles of the AEP proteins DRT.As glândulas salivares são afetadas durante a radioterapia na região da cabeça e pescoço, levando à redução do fluxo salivar e alterando a composição da saliva. Nesta tese, composta de 6 artigos, avaliamos as alterações no perfil proteômico da película adquirida do esmalte (PAE) e da saliva em pacientes com câncer de cabeça e pescoço (CCP) submetidos à radioterapia, para procurar estratégias terapêuticas e biomarcadores prognósticos. Nos primeiros 2 artigos, os protocolos para padronização da análise proteômica da saliva e coleta da PAE são descritos. Nos artigos 3, 4 e 5, pacientes com CCP tiveram suas PAEs e salivas coletados antes (BRT), durante (2-5 semanas; DRT) e após (3-4 meses; ART) radioterapia. As salivas e PAEs também foram coletadas de pacientes saudáveis (controle; C). As proteínas foram extraídas e processadas para proteômica. Os fluxos salivares também foram avaliados. No total, 1.055 proteínas foram identificadas na saliva não-estimulada, sendo 47 comuns a todos os grupos, enquanto 86, 86, 286 e 395 foram encontradas exclusivamente C, BRT, DRT e ART, respectivamente. Notavelmente, alfa-enolase foi aumentada 35X DRT em comparação com BRT, enquanto proteínas ricas em prolina (PRPs) diminuíram. ART houve aumento de 16X da scaffold attachment factor-B1 e diminuição de 3X da alfa-enolase e cistatinas. Em comparação ao C, nos pacientes BRT houve aumento de cistatina-C, lisozima C, histatina-1 e PRPs na saliva (artigo 3). Diferenças significativas foram observadas entre fluxos salivares estimulados e não-estimulados para C e BRT (p>0,001), mas não para DRT e ART. Proteínas envolvidas com apoptose e resistência antibacteriana foram diminuídas na saliva estimulada comparada com a não-estimulada DRT e ART (artigo 4). Na PAE, Statherin aumentou mais de 9X e hemoglobinas aumentaram mais de 5X DRT comparado com BRT, enquanto lactotransferrina, proteínas ricas em prolina, cistatinas, neutrófilo-defensinas e histatina-1 diminuíram. ART, lactotransferrina e histonas aumentaram, porém Statherin e alfa-amilases diminuíram. MOAP-1 foi encontrada exclusivamente ART comparada com BRT. Quando comparado ao Controle, PAE dos pacientes BRT apresentou aumento das proteínas relacionadas à percepção do sabor amargo, mucina-7 e alfa-amilases, enquanto cistatina-S diminuiu (artigo 5). No artigo 6, avaliou-se o efeito protetor da PAE formada in vitro no esmalte por diferentes tempos, bem como sua engenharia com peptídeo da estaterina (StatpSpS), contra erosão inicial. A PAE protegeu quase instantaneamente esmalte natural contra a erosão, mesmo com tempos de formação tão curtos quanto 1 min. Tempos de formação mais longos não aumentaram a proteção. O StatpSpS sozinho conferiu proteção similar àquela da PAE. Em conclusão, tanto o CCP quanto a radioterapia alteram profundamente o proteoma da saliva e da PAE. O fluxo salivar não-estimulado é a melhor alternativa na busca por biomarcadores. Monitorar níveis de alfa-enolase na saliva não-estimulada DRT e MOAP-1 na PAE ART podem ser estratégias possíveis para predizer a eficácia da radioterapia. Nossos resultados fornecem importantes informações para o desenvolvimento de produtos odontológicos mais eficazes para estes pacientes e contribuem para um melhor entendimento do papel protetor da PAE e das alterações progressivas que ocorrem nas proteínas salivares em consequência da radioterapia

Label-Free Quantitative Proteomic Analysis Reveals Inflammatory Pattern Associated with Obesity and Periodontitis in Pregnant Women

Obesity and pregnancy may have synergistic effects on periodontal condition, and proteomics could be an ideal approach to highlight the pathophysiological mechanisms associated with these outcomes. This study analyzed the salivary proteomics related to obesity and periodontitis in women during pregnancy (T1) and after delivery (T2). Initially, 126 women were recruited and forty were allocated into groups: with obesity and periodontitis (OP); with obesity, but without periodontitis (OWP); with normal BMI, but with periodontitis (NP); with normal BMI and without periodontitis (NWP). Whole-mouth saliva was collected in T1 and T2, and proteins were extracted and individually processed by label-free proteomics (nLC-ESI-MS/MS). The up-regulations of Heat shock 70 kDa protein 1A, 1B, and 1-like were related to both obesity and periodontitis, separately. Albumin and Thioredoxin were up-regulated in periodontitis cases, while Cystatins (mainly S, SA, SN) and Lactotransferrin were down-regulated. The high abundances of Submaxillary gland androgen-regulated protein 3B, Protein S100-A8, Matrix metalloproteinase-9, Heat shock 70 kDa protein 2 and 6, Putative Heat shock 70 kDa protein 7, Heat shock 71 kDa protein, Haptoglobin and Plastin-1 were significant in the combination of obesity and periodontitis. Obesity and periodontitis remarkably altered the proteome of the saliva during pregnancy with substantial alterations after delivery

New insights into the protective effect of statherin-derived peptide for different acquired enamel pellicle formation times on the native human enamel surfaces.

OBJECTIVES This study evaluated the protective impact of acquired enamel pellicle (AEP) engineering with statherin-derived peptide (StatpSpS), considering different AEP formation times. DESIGN A total of 120 native human enamel specimens were divided into 2 main groups: 1) No AEP engineering and 2) AEP engineering with StatpSpS (pretreatment for 1 min; 37 °C, under agitation). Each group was further divided into 4 subgroups: No pellicle, or 1, 60-and-120 min AEP formation times (human saliva; 37 °C). The specimens were then subjected to an erosive challenge (1% citric acid; pH 3.6; 1 min; 25 °C). This procedure was repeated for 5 cycles. Relative surface reflection intensity (%SRI) was measured and scanning electron microscopy (SEM) of the enamel surface was done. RESULTS All AEP engineering groups protected against initial dental erosion in comparison with No pellicle (p  0.05). Regarding the SEM analysis, in the "No AEP engineering & No AEP" group, a more severe effect of citric acid was observed, with more enamel prism heads and scratches on the surface when compared with the other groups. CONCLUSIONS AEP provides almost instant protection at formation times even as short as 1 min, protecting the native enamel against erosion. Treatment with StatpSpS by itself provides similar protection as the AEP

Use of Reflectometer Optipen to assess the preventive effect of a sugarcane cystatin on initial dental erosion in vivo.

The sugarcane cystatin 5 (CaneCPI-5) showed protection against erosion and erosive tooth wear (ETW) under several protocols. However, evaluating these conditions in vivo is hard due to the lack of a suitable device. The aim of this study was to use clinically the relative surface reflection intensity (%SRI) by the Reflectometer Optipen to assess the acquired pellicle engineering with CaneCPI-5 rinse for the prevention of initial erosion in vivo. Nine volunteers were distributed in three cross-over phases, according to the rinse used, as follows: 1) Deionized water (negative control); 2) Elmex® (800 ppm Sn2+, 500 ppm F-; positive control); 3) 0.1 mg/mL CaneCPI-5. The following experimental steps were performed: Initially, the volunteers received prophylaxis and the initial %SRI was performed. Subsequently, they rinsed with the solutions (10 mL; 1min), followed by the formation of the acquired enamel pellicle (AEP; 120min). After, the erosive challenge with citric acid 1%, pH 2.5 (10 μL; 10s) was performed (in isolation) on the buccal surface of the maxillary central incisors (right and left). The calcium present in the acid was analyzed by Arsenazo III method. Finally, the final %SRI was performed. Data were analyzed by Kruskal-Wallis/Dunn's tests and Spearman's correlation were used (p < 0.05). For both variables, the negative control led to significantly less protection (lower reflectivity and higher calcium release) in comparison with the other groups. The best protection (higher reflectivity and lower calcium release) was observed for the Elmex® and the CaneCPI-5 groups, with no significant differences between them (p < 0.05). There was a significant correlation between both analyzes. The Reflectometer Optipen demonstrated to be a good device to assess clinically. Moreover, CaneCPI-5 rinse proved effective through acquired pellicle engineering against initial erosion in vivo